RESUMEN
Myelin oligodendrocyte glycoprotein (MOG) is an Ag present in the myelin sheath of the CNS thought to be targeted by the autoimmune T cell response in multiple sclerosis (MS). In this study, we have for the first time characterized the T cell epitopes of human MOG restricted by HLA-DR4 (DRB1*0401), an MHC class II allele associated with MS in a subpopulation of patients. Using MHC binding algorithms, we have predicted MOG peptide binding to HLA-DR4 (DRB1*0401) and subsequently defined the in vivo T cell reactivity to overlapping MOG peptides by testing HLA-DR4 (DRB1*0401) transgenic mice immunized with recombinant human (rh)MOG. The data indicated that MOG peptide 97-108 (core 99-107, FFRDHSYQE) was the immunodominant HLA-DR4-restricted T cell epitope in vivo. This peptide has a high in vitro binding affinity for HLA-DR4 (DRB1*0401) and upon immunization induced severe experimental autoimmune encephalomyelitis in the HLA-DR4 transgenic mice. Interestingly, the same peptide was presented by human B cells expressing HLA-DR4 (DRB1*0401), suggesting a role for the identified MOG epitopes in the pathogenesis of human MS.
Asunto(s)
Presentación de Antígeno , Linfocitos B/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/genética , Esclerosis Múltiple/inmunología , Glicoproteína Asociada a Mielina/inmunología , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Autoantígenos/inmunología , Autoantígenos/metabolismo , Unión Competitiva , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/patología , Mapeo Epitopo , Epítopos de Linfocito T/metabolismo , Antígenos HLA-DR/metabolismo , Cadenas HLA-DRB1 , Humanos , Epítopos Inmunodominantes , Cinética , Ratones , Ratones Transgénicos , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/química , Glicoproteína Asociada a Mielina/metabolismo , Glicoproteína Asociada a Mielina/farmacología , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacologíaRESUMEN
The effects of rabbit-derived polyclonal Ab against PcrV, a protein involved in the translocation of type III secreted toxins of Pseudomonas aeruginosa, was investigated in two animal models of P. aeruginosa sepsis. In a mouse survival study, the i.v. administration of anti-PcrV IgG after the airspace instillation of a lethal dose of P. aeruginosa resulted in the complete survival of the animals. In a rabbit model of septic shock associated with Pseudomonas-induced lung injury, animals treated with anti-PcrV IgG intratracheally or i.v. had significant decreases in lung injury, bacteremia, and plasma TNF-alpha and significant improvement in the hemodynamic parameters associated with shock compared with animals treated in a similar manner with nonspecific control IgG. The administration of anti-PcrV F(ab')(2) showed protective effects comparable to those of whole anti-PcrV IgG. These results document that the therapeutic administration of anti-PcrV IgG blocks the type III secretion system-mediated virulence of P. aeruginosa and prevents septic shock and death, and that these protective effects are largely Fc independent. We conclude that Ab therapy neutralizing the type III secretion system has significant potential against lethal P. aeruginosa infections.
Asunto(s)
Anticuerpos Antibacterianos/uso terapéutico , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Infecciones por Pseudomonas/terapia , Pseudomonas aeruginosa/inmunología , Sepsis/terapia , Animales , Anticuerpos Antibacterianos/metabolismo , Bacteriemia/microbiología , Bacteriemia/fisiopatología , Bacteriemia/terapia , Hemodinámica , Inmunización Pasiva , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Inmunoglobulina G/uso terapéutico , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas Citotóxicas Formadoras de Poros , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/patogenicidad , Conejos , Sepsis/microbiología , Sepsis/fisiopatología , Choque Séptico/microbiología , Choque Séptico/fisiopatología , Choque Séptico/terapia , Tasa de SupervivenciaRESUMEN
HLA DR3 is an MHC molecule that reportedly predisposes humans to myasthenia gravis (MG). Though MG is an Ab-mediated autoimmune disease, CD4+ T cells are essential for the generation of high-affinity Abs; hence the specificities of autoreactive CD4+ T cells are important. In this study we report the HLA DR3-restricted T cell determinants on the extracellular region sequence of human acetylcholine receptor alpha subunit. We find two promiscuous determinants on this region 141-160 and 171-190 as defined by their immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in the absence of endogenous mouse class II molecules. We also studied the minimal determinants of these two regions by truncation analysis, and the MHC binding affinity of a set of overlapping peptides spanning the complete sequence region of human acetylcholine receptor alpha subunit. One of the peptide sequences strongly immunogenic in HLA DR3-transgenic mice also had the highest binding affinity to HLA DR3. Identification of T cell determinants restricted to an MHC molecule known to predispose to MG may be an important step toward the development of peptide-based immunomodulation strategies for this autoimmune disease.
Asunto(s)
Antígeno HLA-DR3/genética , Fragmentos de Péptidos/metabolismo , Receptores Colinérgicos/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Predisposición Genética a la Enfermedad , Antígeno HLA-DR3/metabolismo , Humanos , Epítopos Inmunodominantes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Datos de Secuencia Molecular , Miastenia Gravis Autoinmune Experimental/genética , Miastenia Gravis Autoinmune Experimental/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Receptores Colinérgicos/genética , Receptores Colinérgicos/inmunología , Eliminación de SecuenciaRESUMEN
Efforts to deliver therapeutic genes are frequently rebuffed by the body's adaptive immune response against viral delivery vectors. Attempts to circumvent this problem using non-viral delivery systems have encountered problems with transient expression and inflammatory responses induced by reaction of the innate immune system reacting against bacterial DNA. However, within the past decade, these barriers to non-viral DNA delivery have been recognized as potential allies in the development of novel vaccines for cancer and infectious disease. This review summarizes preclinical and current clinical studies testing the formulation, delivery route and adjuvant options in the development of novel DNA-based vaccines.
Asunto(s)
Antígenos de Histocompatibilidad/uso terapéutico , Inmunoterapia/tendencias , Complejo Mayor de Histocompatibilidad/inmunología , Animales , Enfermedades Autoinmunes/terapia , Productos del Gen gag/metabolismo , Productos del Gen tax/metabolismo , Antígeno HLA-A2 , Virus Linfotrópico T Tipo 1 Humano , Humanos , Inmunoglobulina G/metabolismo , Ratones , Paraparesia Espástica Tropical/diagnóstico , Paraparesia Espástica Tropical/virología , Linfocitos T/inmunologíaRESUMEN
A recurring epitope in the human acetylcholine receptor (AChR) alpha subunit (alpha146-160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101-"DR52a"-or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic "anchor" residues at peptide p1 and p9; also of Asp at p4, which complements this allele's distinctive Arg74 in DRbeta. Surprisingly, despite the 9 sequence differences in DRbeta between DR52a and DR3, merely reducing the bulk of the peptide's p1 anchor residue (Trp149-->Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the alpha helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping alpha140-160 region peptides (in proliferation assays). Even though both use Vbeta2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact-a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.
Asunto(s)
Presentación de Antígeno , Epítopos de Linfocito B/metabolismo , Epítopos de Linfocito T/metabolismo , Antígenos HLA-DR/inmunología , Receptores Colinérgicos/inmunología , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/inmunología , Animales , Línea Celular , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Miastenia Gravis/inmunología , Péptidos/síntesis química , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , TorpedoAsunto(s)
Miastenia Gravis/inmunología , Miastenia Gravis/patología , Receptores Colinérgicos/inmunología , Linfocitos T/inmunología , Timoma/inmunología , Timo/patología , Neoplasias del Timo/inmunología , Secuencia de Aminoácidos , Animales , Variación Genética , Antígenos HLA-DP/inmunología , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Receptores Colinérgicos/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Timectomía , Timoma/cirugía , Timo/inmunología , Neoplasias del Timo/patologíaRESUMEN
In rheumatoid arthritis, HLA-DR alleles associated with elevated relative risk share a common sequence in the third hypervariable domain of the major histocompatibility complex class II molecule (MHC II). Immunization of mice with a peptide vaccine comprised of the appropriate MHC II sequence in adjuvant blocked the onset and reduced the relapse rate of experimental autoimmune encephalomyelitis (EAE). A phase I clinical trial testing a single injection of a third hypervariable domain peptide from the HLA-DRB1*0401 sequence in alum adjuvant showed that the vaccine is well tolerated and generates an anti-HLA-DR antibody response in approximately 25% of the treated patients. A phase II trial testing multiple boosts is in progress. In a more antigen-specific approach, solubilized MHC II molecules loaded with an autoantigenic peptide are injected intravenously to induce unresponsiveness by the binding of the T-cell receptor (TCR) in the absence of costimulation. Appropriate soluble MHC II:autoantigenic peptide complexes inhibit the recall antigen proliferative response of T clones or draining lymph node cells, and reduce the progression of EAE and experimental autoimmune myasthenia gravis (EAMG). A test of a soluble HLA-DR2:myelin basic protein (MBP) complex in multiple sclerosis is progressing in phase I.
Asunto(s)
Presentación de Antígeno/inmunología , Enfermedades Autoinmunes/terapia , Antígenos de Histocompatibilidad Clase II/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Humanos , Esclerosis Múltiple/inmunología , Péptidos/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
We extended the sensitivity of the ELISPOT assay by including an antigen-driven proliferation step prior to a final restimulation with antigen and irradiated antigen presenting cells (APCs). This improved sensitivity made the modified ELISPOT assay better suited to the detection of rare or low frequency T lymphocytes than the standard ELISPOT assay or alternatives such as limiting dilution analysis or in situ hybridization. Use of ELISA-grade plastic or polyvinylidene difluoride (PVDF) plates for the detection of different cytokines improved the signal-to-noise ratio for counting cytokine spots, and use of video computer imaging software improved objective quantitation. Analysis of antigen-reactive peripheral blood mononuclear cells (PBMC) from multiple sclerosis (MS) patients using both the traditional and our modified ELISPOT assay demonstrate a > 10-fold increase in numbers of myelin basic protein (MBP)-responsive T cells detected (an average of less than 1 spot forming cell (SFC) per 2 x 10(5) PBMC with the standard assay compared to 19 SFC per 2 x 10(5) PBMC with the modified assay). In addition, the modified ELISPOT assay could be performed with frozen PBMC, which permitted greater flexibility in sample processing, multiple use of a single sample as an internal standard, and simultaneous analysis of samples collected at different time points. This modified ELISPOT assay has many applications, including analysis of cytokine profiles in rare T cell populations, identification of antigen-responsive individuals as PBMC donors for T lymphocyte cloning or for therapeutic intervention, and assessment of vaccine or therapeutic efficacy as a surrogate clinical marker.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Linfocitos T/inmunología , Secuencia de Aminoácidos , Humanos , Interleucina-2/farmacología , Linfocinas/análisis , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
Within the past year a host of antigen-specific therapies for multiple sclerosis (MS) progressed along the path from IND submission to FDA approval. The Immune Response Corporation vaccinated patients with a Vbeta6 peptide, demonstrating that the vaccine was immunogenic, well tolerated, and reduced the number of Vbeta6+ T-cells in the cerebrospinal fluid (CSF). Connetics Corp. conducted a Phase I/II trial on chronic progressive MS patients vaccinated with CDR2 peptides from TCR Vbeta55.2 and found that patients with a measurable response to the vaccine remained clinically stable for a year. A study at the University of Alberta MS Patient Care and Research Clinic demonstrated that intrathecal injection of a B cell/T cell epitope of myelin basic protein (MBP) decreased the level of antiMBP antibody, but iv. administration did not decrease the relapse rate. AutoImmune Inc. completed a Phase III trial of oral myelin in the spring of 1997 which failed to show a statistical difference between those patients fed placebo and those fed daily capsules of myelin protein (Myoral). Three phase I trials of iv. myelin antigen(s) were initiated: MP4 (Alexion Pharmaceuticals, Inc.), a recombinant fusion of myelin basic protein and proteolipid protein; AG284 (Anergen, Inc.), a solubilised HLA-DR2:MBP peptide complex; and NBI-5788 (Neurocrine Biosciences, Inc.), an altered peptide ligand of an immunodominant MBP T-cell epitope. Following the conclusion of a successful Phase III clinical trial, TEVA Pharmaceutical Industries LTD received FDA approval to market Copaxone (glatiramer acetate) for the treatment of relapsing-remitting MS in December of 1996 and launched the product in 1997. The recent preclinical research and clinical trial status of these antigen-specific MS therapeutics are summarized in this review.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos/administración & dosificación , Animales , Biotecnología , Pollos , Emulsiones , Inmunización , Inyecciones Subcutáneas , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Sonicación , Linfocitos T/inmunologíaRESUMEN
Stimulation of T lymphocytes through the T cell receptor in the absence of costimulatory signal(s) induces a state of unresponsiveness to subsequent antigen presentation. We have employed solubilized complexes consisting of rat class II MHC molecules containing an immunodominant peptide of the acetylcholine receptor (AChR alpha 100-116) to induce unresponsiveness in the autoreactive T lymphocytes mediating an animal model of myasthenia gravis. In vitro incubation of rat T cell lines specific for peptide AChR alpha 100-116 with solubilized complexes of MHC II and AChR alpha 100-116 (MHC II:AChR alpha 100-116) rendered the T cells unresponsive to subsequent stimulation by antigen presenting cells and the peptide. T cell lines with a broader specificity to the entire AChR protein pentamer had an 81% reduction in proliferation to AChR following a preincubation with solubilized MHC II:AChR alpha 100-116. Treatment with the solubilized MHC II:AChR alpha 100-116 induced phosphatidylinositol 4,5-bisphosphate hydrolysis, an early signalling event associated with binding to the TCR. Rats primed with AChR and injected intravenously with MHC II:AChR alpha 100-116 had reduced in vitro T cell proliferation to the AChR alpha 100-116 peptide and to whole AChR. Solubilized MHC II:AChR alpha 100-116 injected i.v. into rats exhibiting serological clinical symptoms of experimental autoimmune myasthenia gravis (EAMG) prevented death in 67% of the treated animals, compared to a 0-20% survival rate in all other control groups. These results demonstrate that solubilized MHC II complexed with an immunodominant autoantigenic peptide is tolerogenic and improves the survival rate of rats with EAMG, suggesting the basis for an antigen-specific therapy in autoimmune diseases such as MG.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/inmunología , Tolerancia Inmunológica/inmunología , Miastenia Gravis/inmunología , Receptores Colinérgicos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , División Celular , Línea Celular , Modelos Animales de Enfermedad , Inositol 1,4,5-Trifosfato/inmunología , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Linfocitos T/citologíaRESUMEN
Using fluorescence-activated cell sorting combined with fluorescence microscopy the mechanism of embryonic germ cell death in the mouse has been shown to be apoptosis. Primordial germ cells (PGCs) from embryos at specific developmental stages have been analyzed, and cells with apoptotic morphology have been isolated by cell sorting. In the female, apoptotic oogonia at Day 13 and apoptotic oocytes at Days 15 and 17 were found. In the male, apoptotic cells were seen on Day 13 through Day 17. Apoptotic germ cells were not detected at Day 12 (combined male and female PGCs). Examination of sorted cells by fluorescence microscopy and by light microscopic analysis after alkaline phosphatase staining confirmed that the cells are apoptotic germ cells. Electron microscopy further confirmed that cells showing the morphological characteristics of apoptosis are present.
Asunto(s)
Apoptosis , Ratones Endogámicos BALB C/embriología , Oocitos/citología , Oogonios/citología , Espermatogonias/citología , Animales , Ciclo Celular , Femenino , Citometría de Flujo , Edad Gestacional , Luz , Masculino , Ratones , Dispersión de RadiaciónRESUMEN
As an approach to mapping replicons in an extended chromosomal region, the temporal order of DNA replication was analyzed in the murine major histocompatibility gene complex (MHC). Replicating DNA from T-lymphoma and myelomonocyte cell lines was density labeled with bromodeoxyuridine and extracted from cells which had been fractionated into different stages of S phase by centrifugal elutriation. The replicating DNA from each fraction of S phase was separated from nonreplicating DNA on density gradients, blotted, and hybridized with 34 specific MHC probes. The earliest replication occurred in the vicinity of transcribed genes K, HAM1 and HAM2, RD, B144, D, L, T18, and T3. The temporal order of replication of groups of DNA segments suggests the location of five or six replicons within the H-2 complex, some of which appear to be either unidirectional or markedly asymmetric. The rates of replication through each of these apparent replicons appear to be similar. The TL region of the S49.1 T-lymphoma cells, which contains at least three transcribed genes, replicates earlier than the inactive TL region of WEHI-3 myelomonocytic cells. These results provide further evidence of a relationship between transcription and the initiation of DNA replication in mammalian cells. The mouse MHC examined in this study is the largest chromosomal region (> 2,000 kb) measured for timing of replication to date.
Asunto(s)
Replicación del ADN , Antígenos H-2/genética , Complejo Mayor de Histocompatibilidad , Animales , Línea Celular , Ratones , Células Tumorales CultivadasRESUMEN
Rhodamine absorbed to protein was removed from rhodamine-conjugated antibody solutions by adsorption to hydrophobic macroporous beads (Bio-Beads SM-2) following gel permeation chromatography. This simple technique eliminated the contaminating free dye more effectively than gel filtration alone, but neither significantly reduced the level of fluorescently conjugated antibody nor altered the latter's binding characteristics. Passage of the fluorescent antibody solution over the SM-2 beads eliminated the high background of nonspecific staining caused by internalization of residual free dye.